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Known to his friends as "Stew", Witwer grew up in Glenview, Illinois, a small suburb town of Chicago. There, he attended Glenbrook South High School and became involved in theater as well as becoming the lead singer in a band called "Love Plumber". After receiving marks that were less than satisfactory, Witwer later attended the Juilliard School for drama stating that it was the only school that accepted him on the fact that they didn't look at his transcripts.

In 2003, Witwer made a appearance in Star Trek: Enterprise in the episode "The Shipment" as a Xindi Arboreal technician.

From 2008 to 2009, Witwer had been cast as Davis Bloome for the eighth season of Smallville.[1] His character was a charming paramedic that doubles as the show's incarnation of Doomsday. Although uncredited, he also portrayed Major Zod in the last scene of the Season eight finale while the character would be portrayed by British actor Callum Blue for the duration of the show's ninth season.

In 2010 he starred as a walker in the pilot episode of AMC's The Walking Dead.

From 2011 to 2014, Witwer portrayed the lead vampire Aidan in the Syfy Channel's remake of the BBC original series Being Human.

From 2016, he portrays Mr. Hyde from the sixth season in the ABC series Once Upon a Time.

When Amy Beth Christenson's original concept art of Galen Marek looked strikingly like Sam Witwer, the vice president at the time noted the resemblance due to recognizing Witwer for Battlestar Galactica so he was contacted to audition.[2] From there, he became the complete basis for the main character's voice and appearance in Star Wars: The Force Unleashed. His likeness was created through motion capture for the game to build the character's appearance and range of facial expressions. Witwer's performance was recorded on HD cameras (as were all the actors who worked on the game). While the game was in production, Witwer only knew his character as "Starkiller".[3] Witwer also voiced Galen Marek (known only as "The Apprentice") in Soulcalibur IV. Witwer returned to play Starkiller in the sequel Star Wars: The Force Unleashed II. For this character, he served in the same capacity as before by providing his voice and likeness to the main character.[4] Witwer also voiced Palpatine in Star Wars: The Force Unleashed, and gave his voice and likeness to the imperfect genetic replicas and the Dark Apprentice in Star Wars: The Force Unleashed II.[4]